Method for enrichment of natural antisense messenger RNA

ABSTRACT

A method for enrichment of natural antisense mRNA which involves hybridization of cDNA obtained from sense RNA with cDNA obtained from antisense RNA, followed by DNA polymerase treatment of the sense-antisense hybrid DNA molecule. A natural antisense library can be generated by cloning of sense-antisense hybrid DNA molecules in a vector.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of application Ser.No. 09/680,420, filed Oct. 6, 2000, now U.S. Pat. No 6,528,262, whichclaims priority under 35 U.S.C. §119(e) from U.S. provisionalapplication No. 60/157,843, filed Oct. 6, 1999, the entire contents ofboth applications being hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for enriching antisensemessenger RNA that are naturally expressed in cells.

2. Description of the Related Art

The functional information of the genes in the genome is unidirectional.Taking as an example the most simple gene which encodes a protein, theRNAs encoding a protein are transcribed from one of the strands anddefine one mRNA that possesses the information for the translation of asingle protein. This simplistic view is obviously more complex for manygenes due to mechanisms such as alternative splicing and intron encodedmRNAs. While this general view of the unidirectional nature of genes istrue with regard to structural information, it is not so with regard toregulatory information. The possibility of transcription of the otherstrand of a gene, in the reverse direction, will result in theproduction of an RNA that is complementary to the mRNA that possessesthe structural information (the sense mRNA). Such RNAs, termed antisenseRNAs, have the potential to strongly bind to the sense mRNA, producing adouble stranded RNA, and to inhibit the realization of the structuralinformation. However, it is possible that these RNAs also possessstructural information.

In general, natural antisense RNAs are endogenous transcripts containingregions complementary to transcripts of other genes or other transcriptsarising from the same gene locus. Antisense RNA is an RNA which containsa stretch of nucleotides complementary to another RNA that has somecellular function. The length of the complementary stretch is usually afew hundred nucleotides, but shorter stretches can also be important.The expression of antisense RNA is a powerful way of regulating thebiological function of the sense RNA molecules. Natural antisense RNAshave been shown to play important regulatory roles, including control ofcell growth, malignant transformation and other cellular phenotypes.Through the formation of a stable duplex between the sense RNA andantisense RNA, the normal or sense RNA transcript can be renderedinactive and untranslatable.

Natural antisense transcripts can either be cis-encoded ortrans-encoded. Cis-encoded antisense arises from transcription of thecomplementary strand of the sense gene; both sense and antisensetranscripts originate from the same locus and thus, the antisensetranscripts have regions of perfect complementarity to the sense mRNA.Trans-encoded antisense arises from transcription of a different genomiclocus, and accordingly, it is expected that in such cases thecomplementarity of the antisense region will be not complete.

Following the discovery of natural antisense RNAs in prokaryotes,natural antisense RNAs were also discovered in a variety of eukaryotescovering a wide range of the phylogenetic tree, including viruses(Michael et al., 1994), slime molds (Lee et al., 1993), insects(Lankenau et al., 1994), amphibians (Kimelman et al., 1989), birds(Farrell et al., 1995) and mammals (Murphy et al., 1994). Moreover, mostof the genes for which endogenous antisense transcripts were discoveredencode proteins of key regulatory roles in important cellularphenotypes, such as cellular proliferation (Chang et al., 1991),apoptosis (Khochbin et al., 1989) and embryonic development (Bedford etal., 1995), or of key cellular processes such as translation (Noguchi etal., 1994), transcription (Krystal et al., 1990) and splicing (Fu etal., 1992). The thorough review article by Vanhee-Brossollet and Vaquero(1998) offer a summary of all antisense RNAs discovered so far and theirfunctional importance. The evidence suggests that control of geneexpression by endogenous antisense RNAs is one of the regulatorymechanisms in the cell and is widespread throughout the eukaryotickingdom.

All of the antisense transcripts discovered so far were found bysporadic experiment stemming from studies of single genes. This impliesthat the antisense regulatory mechanism might be of general importanceand relevant for many more genes. Thus, a general method for findingthose mRNAs in the cell for which an antisense RNA exists would be ofgreat value. The object of the approach and method of the presentinvention is to enable those in the art to investigate which mRNAs inthe cell have antisense RNAs, as compared to studies done up until nowwhich investigated the question “does this specific mRNA have anantisense RNA counterpart?”

Citation of any document herein is not intended as an admission thatsuch document is pertinent prior art, or considered material to thepatentability of any claim of the present application. Any statement asto content or a date of any document is based on the informationavailable to applicant at the time of filing and does not constitute anadmission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention is directed to rapid methods for enrichment ofnatural antisense mRNA (the exonuclease and polymerase activities of DNApolymerases on the sense-antisense double-stranded hybrid), followed byamplification and cloning of its corresponding cDNA. Thus, the inventionovercomes the deficiencies in the prior art as discussed above. Thesemethods provide for the enrichment and detection of natural antisensemRNA from any natural source of RNA. Poly A+ mRNA in a sample of RNA isconverted into single-stranded cDNA which is denatured to disrupt anysecondary structure, and then allowed to re-anneal under stringentconditions with any other cDNA having a segment with a significantcomplementary sequence to form a hybrid molecule with a double-strandedcDNA segment. Sense and antisense cDNAs hybridize to each other and formdouble-stranded stretches or segments of DNA. In an embodiment of thepresent invention, the resulting hybridization products withdouble-stranded DNA segments are treated with a DNA polymerase, such asT4 DNA polymerase, which has a 5′ to 3′ polymerase activity and a 3′ to5′ exonuclease activity. The resulting double-stranded molecules arethen amplified and cloned.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic presentation of an embodiment of the naturalantisense enrichment procedure according to the present invention.

FIG. 2 shows an agarose gel electrophoresis analysis of PCRamplification products generated from single stranded cDNA obtained fromreverse transcription (RT) of c-erb, Ku autoantigen, and GAPDH beforeand after the antisense enrichment procedure according to FIG. 1.

FIG. 3 shows gene-specific RT-PCR analysis of the expression of sense(S) and antisense (AS) mRNAs of clone N1-15 by use of specific primerscomplementary to the two strands, and GAPDH control by agarose gelelectrophoresis. Clone N1-15 (RbAp48 mRNA encoding retinoblastomabinding protein, GenBank accession number X74262) was obtained from theantisense enrichment procedure. The lane designated M is a lane ofmolecular weight markers.

FIG. 4 shows a Northern blot analysis of RbAp48 mRNAs with sense andantisense RbAp48 single stranded probes.

FIG. 5 shows a schematic presentation of a second embodiment of thenatural antisense enrichment procedure according to the presentinvention.

FIG. 6 shows a schematic presentation of a variation of the secondembodiment shown in FIG. 5, where a 3′-end of the B-ssc DNA is blocked.

FIG. 7 shows a schematic presentation of hybrids generated that areresistant to the antisense enrichment procedure according to the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the development of simple and rapidmethods for the enrichment of natural antisense mRNA from any source ofmRNA. The source of the poly A+ mRNA used in the present method can be acell line, tissue, whole organism, or even a mixture of poly A+ mRNAfrom two or more sources. The source(s) of mRNA is preferably a naturalsource(s). A mixture of RNA from different sources can be, for example,a mix of control mRNA derived from normal tissue or cells and mRNA fromtreated tissues or cells, or a mix of control mRNA and mRNA derived frompathologic tissue mRNA, etc.

A method for enrichment of natural antisense messenger RNA (mRNA)according to the present invention is schematically presented in FIG. 1.A population of single-stranded cDNA molecules is generated from asource of poly A⁺ RNA (RNA recovered from oligo dT columns after loadingthereon a sample of total RNA from cells or tissue), where such columnsbind the mRNA that contain a polyA stretch. The vast majority ofeukaryotic mRNA have polyA tails at their 3′-end. The RNA that does notcontain polyA is washed away and the bound RNA is recovered. The resultis an RNA sample enriched for the polyA-containing mRNAs. The polyA⁺ RNAis used as starting mRNA template for reverse transcription from anoligonucleotide primer. The oligonucleotide primer containspolydeoxythymidine (oligo dT) which can anneal to the poly A+ tail ofmRNA, and also preferably contains one or more restriction enzymecleavage sites for later use in cloning into vectors, and a further“END” sequence at the 5′-end of the primer that allows specificamplification.

The population of cDNA molecules generated by reverse transcription witha reverse transcriptase is heated to disrupt any secondary structure,such as self-annealing, and then incubated under stringent conditions toallow hybridization of complementary cDNA segments. It is expected thata cDNA (antisense) complementarity to the sense cDNA will form an atleast partially double-stranded hybrid molecule. cDNA generated fromcis-encoded antisense mRNA, which arises from transcription of thecomplementary strand of the sense gene, will have a certain region ofperfect complementarity to its corresponding sense cDNA. However, cDNAgenerated from trans-encoded antisense, which arises from transcriptionof a different genomic locus, is expected to have only partialcomplementarity to a sense cDNA from a different genomic locus. Even insuch a case, functional trans-encoded antisense will generate adouble-stranded structure stable enough to be retained by the enrichmentmethod.

Treating the hybrid molecule, which is at least partiallydouble-stranded, with a DNA polymerase having 5′ to 3′ polymeraseactivity and 3′ to 5′ exonuclease activity produces double-strandedmolecules with complete complementarity. This treatment with DNApolymerase cleaves single-stranded DNA molecules with free 3′ ends untilthe DNA polymerase reaches a region of stable double-stranded structure,producing a DNA molecule with a double-stranded region and one or twoadjacent single-stranded DNA regions with free 5′ ends. This serves as atemplate for the 5′ to 3′ polymerase activity of the enzyme whichproduces a final product with blunt ends. The DNA polymerase having 3′to 5′ polymerase activity useful in the method according to the presentinvention is preferably T4 DNA polymerase. Four other enzymes useful inthe method according to the present invention are Platinum Pfx DNApolymerase and Deep Vent DNA polymerase (both from New England Biolabs,Beverly, Mass.), Pwo DNA polymerase (Roche) and Pfu DNA polymerase(Stratagene, La Jolla, Calif.). Other polymerases can be used, includingbut not limited to any suitable DNA polymerase having both aforesaidactivities.

The double-stranded molecules resulting from the DNA polymerase reactionare amplified by the polymerase chain reaction (PCR) using a polymerase,such as Taq DNA polymerase, or other thermostable polymerases, andpreferably with a primer identical to the END region of the polydT-containing oligonucleotide primer previously used to generate thepopulation of cDNA. Thus, natural antisense mRNA molecules as convertedto double-stranded cDNA molecules are enriched. The amplifieddouble-stranded DNA molecule enriched for natural antisense mRNA can beeasily cloned into a vector using one or more restriction enzymecleavage sites at the ends thereof (i.e., derived from theoligonucleotide primer). Confirmation that the cloned double-strandedcDNA encodes a natural antisense mRNA can be obtained by RT-PCR orNorthern blot analysis as described in the Example 1 herein.

The oligonucleotide primer used for amplifying the double-strandedmolecules from the DNA polymerase reaction is complementary to andpreferably identical to the END sequence located at the 5′-end of thepolydT primer for initially generating the double-stranded molecules.The location of the END sequence before (5′ to) the restriction enzymecleavage site(s) and polydT region) only amplifies the correct templateand gives rise to products that have the restriction enzyme cleavagesite(s) available for cloning. Thus, natural antisense molecules canonly be enriched and amplified if there is a successful polymerizationwhich produces templates that can be amplified by the EndogenousAntisense Identification (EASI) procedure of the present invention.

Because natural antisense RNAs have been shown to play importantregulatory roles or provide new regulatory information for known genesin the control of cell growth, malignant transformation, and othercellular phenotypes, the present method provides a basis for finding newgenes with important cellular regulatory roles or new regulatoryinformation for known genes and provides a starting material fordevelopment of an antisense-based therapeutic to treat a disease ordisorder in which the down-regulation or inhibition of the sense gene ortranscript is sought.

The EASI method allows production of a cDNA population enriched forsequences representing endogenous antisense RNAs. In Example 1, oneapproach to use the method for the identification of the antisense RNAis described. This employs the derivation of cDNA libraries from theEASI enriched cDNA population, followed by sequencing of cDNA clones,characterization of the sequences, and analysis of the existence ofantisense RNA in the cells. Example 2 describes a way to use the EASIenriched cDNA population to derive probes for microarray analysis, whichallows both the detection of antisense transcripts and theirdifferential expression. While the specific RNA polymerase disclosed inExample 2 below is T7 RNA polymerase, this specific RNA polymerase canbe any bacteriophage RNA polymerase which uses DNA as template toproduce RNA, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNApolymerase, etc.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration and is not intended to be limiting ofthe present invention.

EXAMPLE 1

The antisense enrichment procedure according to the present invention,also designated as the Endogenous Antisense Identification (EASI)procedure, was applied to a human glioma cell line and shown inexperiments to enrich for antisense mRNA/cDNA known to have a naturalantisense mRNA. The experimental results and the materials and methodsused in the experiments discussed in this example are provided below.

Materials and Methods

cDNA First Strand Synthesis:

Two μg of poly A+ RNA from human A172 glioma cell line were diluted withdouble distilled water (DDW) up to 6 μl volume. Secondary structureformation or self-annealing of RNA molecules is disrupted by incubationat 80° C. for 2 min and then quick chilled on ice. 4 μl of a 10 mM oligodT (deoxythymidine) primer, which contains cloning/cleavage sites forthe NotI restriction enzyme and has the sequence

-   5′-TTCTAGAATTCAGCGGCCGC(T)₁₈N_(1 (G,A,C))N_(2 (G,A,C,T))-3′ (SEQ ID    NO:1)    (where 5′-TTCTAGAATTCAGCGGCCGC-3′, corresponding to nucleotides 1 to    20 of SEQ ID NO:1, is termed the “END” sequence), 4 μl of 5×    Superscript buffer, 1 μl of RNasin, 2 μl of 0.1M DTT    (dithiothreitol) and 2 μl of 10 mM dNTPs (deoxynucleoside    5′-triphosphates) were added to the poly A+ RNA. After preheating at    42° C., 1 μl of RT Superscript enzyme was added and incubated at    42° C. for 1.5 hours. The reaction mixture was then heated to 80° C.    for 2 min and the RNA strands were digested with alkaline treatment,    followed by neutralization.    Hybridization:

The cDNA generated was precipitated and then resuspended with 30 μlhybridization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, pH 8.0, 0.4 MNaCl, 80% formamide, DDW). Any secondary structure was disrupted at 85°C. for 10 min and then the cDNA was rapidly transferred to a 52° C. bathfor 16 hours. After 16 hours of incubation, the cDNA was precipitatedwith ethanol and resuspended in 30 μl DDW.

T4 DNA Polymerase Treatment:

Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 μl of hybridized cDNA molecules with T4 DNA Polymerase (3-6 units),dNTPs, T4 reaction buffer, and BSA (bovine serum albumin) to a finalvolume of 80 μl. After reacting for 1 hour at 16° C., 5 μl of Klenowsequencing grade enzyme was added and then transferred for 30 min at anincubation temperature of 37° C. The inactivation of the enzymes wascarried out at 75° C. for 10 min, followed by phenol/chloroformextraction and EtOH precipitation. The precipitated pellet wasresuspended in 12 μl of DDW for use as the PCR template in the next PCRamplification step.

Platinum Pfx DNA Polymerase Treatment

Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 1 ml of Platinum Pfx DNApolymerase (2.5 u/ml), 1.5 ml 10 mM dNTPs, 5ml 10× Pfx Amplificationbuffer, 1 ml 50 mM MgSO2 and 11.5 ml DDW to a final volume of 50 ml andthen transferred for incubation at 72° C. for 5 min. The inactivation ofthe enzymes was carried out at 75° C. for 10 min, followed byphenol/chloroform extraction and EtOH precipitation. The precipitatedpellet was resuspended in 12 ml of DDW for use as the PCR template inthe next PCR amplification step.

Deep Vent DNA Polymerase Treatment

Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 2 ml 10 mM dNTPs, 5 ml 10×Thermo polymerase reaction buffer, 2 ml 100 mM MgSO2 and 10 ml DDW to avolume of 49 ml and preincubated at 72° C. for 2 min. 1 ml of Deep VentDNA polymerase (2 u/ml) was added and the whole mixture was transferredfor incubation of the reaction at 72° C. for 5 min. The inactivation ofthe enzymes was carried out at 75° C. for 10 min, followed byphenol/chloroform extraction and EtOH precipitation. The precipitatedpellet was resuspended in 12 ml of DDW for use as the PCR template inthe next PCR amplification step.

Pwo DNA Polymerase Treatment

Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 2 ml 10 mM dNTPs, 5 ml 10×PCRbuffer with 20 mM MgSO2 and 12 ml DDW to a volume of 49 ml and thentransferred for preincubation at 72° C. for 2 min. Then 1 of Pwo DNApolymerase (5 u/ml) was added and the whole mixture was transferred forincubation at 72° C. for 5 min. The inactivation of the enzymes wascarried out at 75° C. for 10 min, followed by phenol/chloroformextraction and EtOH precipitation. The precipitated pellet wasresuspended in 12 ml of DDW for use as the PCR template in the next PCRamplification step.

Pfu DNA Polymerase Treatment

Blunt ending by both removal of single-stranded cDNA 3′ to 5′ andextension of double-strand over a single-stranded template was done on30 ml of hybridized cDNA molecules with 5 ml cloned Pfu DNA polymerase(2.5 u/ml), 5 ml 10 mM dNTPs, 5 ml 10× cloned Pfu polymerase buffer, and5 ml DDW to a final volume of 50 ml and then transferred for incubationat 72° C. for 5 min. The inactivation of the enzymes was carried out at75° C. for 10 min, followed by phenol/chloroform extraction and EtOHprecipitation. The precipitated pellet was resuspended in 12 ml of DDWfor use as the PCR template in the next PCR amplification step.

PCR Amplification:

1 μl from the resuspended DNA pellet resulting from the T4 DNApolymerase treatment was used as a template for PCR amplification withthe same END primer derived from the oligo dT primer used for theinitial cDNA synthesis. The PCR conditions are as follows: The reactionwas performed in a total volume of 50 μl with 1 μl template afterblunting (T4 DNA polymerase treatment), 5 μl Boehringer 10× buffer 2, 1μl 10 mM dNTPs, 4 μl 10 μM primer, 0.5 μl PERFECT MATCH PCR enhancer(Stratagene) La Jolla, Calif., 0.5 μl of (3.5 u/μl) Boehringer Taq highfidelity DNA polymerase (Boehringer-Mannheim) and DDW for the remainingvolume. The temperature program used for PCR was as follows: 2 min of94° C. for denaturation and 16 cycles of: denaturation at 94° C. for 30sec, annealing at 64° C. for 30 sec, and extension at 72° C. for 4 min.At the end of the cycling, the extension was completed at 72° C. for 7min.

Cloning:

The PCR amplified product was digested with NotI and ligated intopBLUESCRIPT vector (Stratagene) linearized with the restriction enzymeEag1. EagI-cleaved sticky ends are compatible with NotI-cleaved stickyends.

The transformants were detected in the presence of X-Gal(5-bromo-4-chloro-3-indolyl β-D-galactopyranoside) and IPTG (isopropylβ-D-thiogalactopyranoside). White colonies containing inserts weresequenced following miniprep DNA purification. All the sequencingproducts were analyzed by common Bioinformatic programs, such as BLAST,and the interesting sequences were used as templates for single-strandedprobes for Northern blot analysis to confirm the presence of anantisense molecule.

A real antisense is determined to be present by use of two reversecomplementary single-strand (ss) probes, each complementary to adifferent strand. For a known gene, the ss probe complementary to thesense strand will hybridize to the sense mRNA and give a signal on aNorthern blot to a band of expected size. The ss probe complementary tothe antisense strand will give a signal on a Northern blot if anantisense mRNA is present in the RNA population.

Reverse Transcription—PCR Amplification

The following gene-specific RT-PCR conditions were found to be of highspecificity to the targeted gene. Poly A+ selected mRNA (100 ng) wasused as template and the reactions were carried out with theThermoscript RT enzyme (100 ng polyA+) (Gibco/BRL). RNA, 4 μl of 5×buffer, 10 μM primer and H₂O were mixed to a final volume of 14 μl. Themixture was incubated at 85° C. for 1 minute and then at 65° C. for 5minutes. The following were then added while the tube was held at 60°C.: 1 μl of 0.1 mM DTT, 1 μl of RNAseOut (40 units/μl), 2 μl of 10 mMdNTPs and 1 μl H₂O. 1 μl of Thermoscript RT (15 units/μl) was added andthe reaction was carried out at 60° C. For 1 hour. The reaction was thenterminated by incubation at 85° C. For 5 minutes with the RNA beingremoved by the addition of 1 μl of RNAseH (1 u/μl; Boehringer) andincubation at 37° C. For 20 minutes.

The PCR amplication reaction was conducted under standard conditionsusing a pair of sense and antisense primers for the tested gene. Taq DNApolymerase from Boehringer was used. The initial cycle consisted ofincubation at 94° C. For 3.5 minutes followed by 5 cycles of:denaturation at 94° C. For 30 seconds, annealing at 62° C. For 30seconds and extension at 72° C. For 2 minutes. This was then followed by25 cycles of: 94° C. For 30 seconds, 58° C. For 30 seconds and 72° C.For 2 minutes. The reaction was terminated with a step of incubation at72° C. For 7 minutes.

Primers and Sequences

The primers and sequences used in the EASI procedure described andexemplified in this example are provided below.

Human c-erb ERB-SEN2: 5′-GATGGGAGTTGTGTGTTTAGTC-3′ (SEQ ID NO:2) ERB-RC:5′-GGAGAGAGAAGTGCAGAGTTCG-3′ (SEQ ID NO:3) Human ku autoantigen KU_FOR:5′-TTAGTACAAACTTAGGGCTCT-3′ (SEQ ID NO:4) KU_REV:5′-TCATGGCAACTCCAGAGCAG-3′ (SEQ ID NO:5) Human GAPDH GAPDH_FOR:5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO:6) GAPDH_REV:5′-TCCACCACCCTGTTGCTGTA-3′ (SEQ ID NO:7) Human RbAp48 (clone N1-15)N1-15FOR: 5′-GGAGTTAGTCCTTGACCACTAG-3′ (SEQ ID NO:8) N1-15RC:5′-GCACTTACACAGTTAGTCATGG-3′ (SEQ ID NO:9) Sequence of clone N1-15-designated SEQ ID NO:10Northern Hybridization Using Single-stranded Probe

For each interesting fragment that was obtained from the bioinformaticanalysis, two primers, forward and reverse, that are suitable for thetwo ends of the fragment were prepared. Northern blots were preparedaccording to CURRENT PROTOCOLS IN MOLECULAR BIOLOGY by F. M. Ausubel etal, Chapter 4.a with 6 μg of poly A+ RNA per lane.

Synthesis of Labeled Single-stranded DNA Probes:

The cDNA insert of the tested gene (isolated by digestion or by PCR fromthe plasmid) was labeled using well-known labeling reactions with Klenowenzyme. A specific sense or antisense primer was used to drive thesynthesis of the probe in the presence of radioactive nucleotides. Thus,only one strand (sense or antisense, depending on the primer used) wassynthesized and labeled with radioactivity. For each gene, the sense andantisense probes were generated and used for hybridization on separateblots.

For the preparation of DNA template, 0.5-1 μg of template from aminiprep DNA isolation procedure was denatured with NaOH treatment andincubated for 5 min. at RT (room temperature), precipitated with NH₄AcpH 5.2, yeast tRNA and 100% EtOH. After ethanol precipitation, thepellet was redissolved in 7 μl of DDW.

For the synthesis of labeled single strands, 50-100 ng of the denaturedtemplate DNA was mixed and annealed with 10 pmols of the appropriateprimer in Klenow buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 7.5 mMDTT), and incubated at two different temperatures, 15 min at 60° C. andthen 15 min. at RT. α-³²P-dCTP (specific activity of 3000 ci/mmol),Klenow enzyme (5 units) and dATP, dTTP and dGTP nucleotide solution (0.5mM final concentration) were then added to the annealing mix andincubated for 20 min. at 37° C. to incorporated label into thesynthesized strand of DNA. Afterwards, “cold” (non-radioactive) dCTP wasadded and the reaction was further incubated for 15 min. at 37° C. Theinactivation of the Klenow enzyme was carried out by incubation at 75°C. For 10 min. The purification of the probe was done on a SEPHADEX G-50column.

Northern Blot Hybridization and Post-hybridization Processing

1-5×10⁶ cpm of denatured labeled probe was added per 1 ml hybridizationsolution and incubated overnight at 65° C.

Prehybridization was carried out for 30 min to 2 hours at 65° C. TheNorthern blot was washed 3 times in 2×SSC, 0.2% SDS for 7 min. at RT andthen twice with high stringency washing solution that contain 0.1×SSC,0.2% SDS for 15 min. at 60° C. The blot was then exposed onto X-Rayfilms.

Results and Discussion

The antisense enrichment procedure was applied to examine the possibleinvolvement of antisense RNAs in the response of the human glioma cellline A172 to hypoxia stress. This is a cell line model for the responseof glial cells in the brain to ischemic injury that can result fromevents such as stoke. To detect antisense mRNAs that are involved in theresponse of the cells to hypoxia conditions, poly A+ selected mRNA wasobtained from cells grown under hypoxia conditions as well as from cellsgrown under normal conditions. Single-stranded cDNA prepared from thetwo mRNA pools were mixed in equal proportions and used as the basis forthe antisense enrichment procedure.

A gene, known to have an endogenous antisense mRNA was compared to twogenes, for which no endogenous antisense mRNA is known to exist, to testthe ability of the antisense enrichment procedure to enrich forantisense cDNAs. The presence of these three genes was tested before andafter application of a preferred embodiment of the antisense enrichmentmethod according to the present invention. As shown in FIG. 2, PCRamplification was performed for each gene with two gene specificprimers, (1) before the antisense enrichment procedure on single strandcDNA (RT), and (2) is known to on DNA obtained after antisenseenrichment (AS). The c-erb cDNA is known to be found at very low levelsin the mRNA and gave no signal after PCR of normal RT products (20 ngpoly A+ selected RNA). However, using a similar amount of DNA template,a strong signal was obtained from DNA obtained from the antisenseenrichment procedure. The two other genes, Ku and GAPDH, for which thereare no known antisense mRNA, and which are expressed at much higherlevels, show a clear decrease in abundance in FIG. 2. This indicatesthat the antisense enrichment procedure according to the presentinvention advantageously enriches for cDNA for which a natural antisensecounterpart exists (c-erb) in contrast to cDNA which lacks a naturalantisense counterpart (Ku, GAPDH).

The final products of the antisense enrichment procedure were cloned asdescribed in the materials and methods section and a library wascreated. After sequencing and bioinformatics analysis, few of the cloneswhich matched known genes were chosen for further analysis to determineif they originated from an endogenous antisense mRNA. In order toexamine the presence of antisense mRNAs to a specific gene, a genespecific RT-PCR method was established in which gene specific primerswere used to initiate the reverse transcription reaction. To detect thenormal (sense) transcript of a gene, an antisense primer was used.Conversely, when a sense primer was used, it should be possible todetect antisense mRNAs. It was found that standard RT-PCR reactions werenot specific enough, and when a gene specific primer, either sense orantisense, was used, amplification products, for which no antisensetranscript was known to exist for the gene, was observed. Moreover,other genes were also amplified. This indicates that standard RT-PCRreaction conditions resulted in non-specific reverse transcription ofother mRNAs. A gene specific RT-PCR procedure was then established usinghighly specific conditions described in the Materials and Methodssection of this example to confirm if the clones originated from naturalantisense RNA. A highly specific RT reaction was performed with a senseprimer (relative to the known sense mRNA) using Thermoscript reversetranscriptase (Gibco BRL).

Under the aforesaid highly specific conditions such a reaction willresult in a PCR product only if an antisense mRNA exists. This wascompared to an RT reaction done using the antisense primer. Table 1includes the list of clones from the antisense enriched library thatwere individually confirmed for the presence of matching antisense RNA.One clone, clone N1-15 (RbAp48 mRNA encoding retinoblastoma bindingprotein, accession number X74262), obtained from the antisenseenrichment-derived library, is shown here in detail. A primer pair(sense and antisense primers) specific for the sequence obtained in thelibrary was synthesized. Each primer was used to derive a RT reactionusing Thermoscript reverse transcriptase. As a control, the same wasdone for GAPDH for which there is no known antisense mRNA. For bothgenes, the sense primer (S) is expected to support reverse transcriptionof antisense mRNA while the antisense primer (AS) is expected to supportthe synthesis of the sense (normal) mRNA. In FIG. 3, a clear signal wasobtained with the sense primer (S) derived RT-PCR of the N1-15 clonewhereas none was obtained (as expected) for the GAPDH mRNA. Theantisense (AS)-derived RT-PCR however gave the expected products. Thisdemonstrates that for clone N1-15, which matches the RbAp48 mRNAencoding retinoblastoma binding protein, PCR products of the expectedsize were obtained for both sense and antisense RT-PCR, whereas thecontrol gene, GAPDH, which does not have any known antisense mRNA,resulted in a product only with the antisense primer. This suggestedthat an endogenous antisense mRNA does exist for this N1-15 gene.

TABLE 1 Strand- Clone Insert specific Northern Blots Bio name Gene nameAccession length Match location % identity RT-PCR Sense Antiseninformatics N1_15 RbAp48 encoding NM_005610 169  2179-2307 100 Positive3.0 Kb 3.5 Kb Positive retinoblastoma binding protein N1_27 CD9 antigenNM_001769 550  621-1171 99 Positive ND ND N1_33 Thymosin b-10 S54005 491  1-447 97 Positive 0.6 Kb none N2_20 Uncharacterized AF187554 170 555-669 100 Negative* 7.0 Kb 0.5 Kb N2_66 2-19 gene (downstream toX55448 206 42663-42866 99 Positive G6PD) N3_13 Calumein AF013759 143 2273-2415 100 Positive ND ND D1-1 SMRTE, Silencing AF125672 111 8573-8667 96 Positive 8.5 kb 3 kb mediator of retinoic acid and thyroidhormone receptor a D1-19 M-phase phosphoprotein, X98260 100  578-661 98Negative ND ND NO Antisense mpp11 D1-48 Metallothionein 2 X97260 189 225-327 98 Positive ND ND D2-31 Integrin a 3 NM_005501 94  4421-4495 98Positive 4.5 kb 4.5 kb D2-3 S100 calcium-binding NM_002966 161  549-649100 Negative ND ND NO Antisense protein A10 D2-7 Regulator of G-proteinAF030108 106  340-436 100 Negative ND ND NO Antisense signaling 5 (RGS5)D2-20 14-3-3n L20422 50  1524-1557 100 Negative ND ND NO AntisenseN26_3_50 Ribosomal protein S11 X06617 586  538-1 99 Positive ND NDPositive Antisense Human isocitrate U52144 gene dehydrogenase mRNAN2_3_40 Homo sapiens ribosomal NM_001030 363   1-329 (29-357) 99Positive ND ND Positive protein S27 (metallopanstimulin 1) (RPS27)Antisense S24A Human ribosomal M31520  249-353 (116-12) 100 gene proteinS24 mRNA N3_45 UbA52 placental mRNA X56999 563   52-552 99 Positive NDND for Ubiquitin-52 fusion protein 25_3_49 Homo sapiens cDNA AK000501662  573-351: 352-24 95 ND ND ND Positive FLJ20494 fis, clone KAT08547M.musculus mRNA for Y08702 504  449-329: 330-236: 86 neuronal protein15.6  186-104 20_2_33 Homo sapiens actin, beta NM_001101  1326-1622(19-315) 97 Positive ND ND (ACTB) mRNA Matches  1688-1791 to normal mRNAand (382-485) to pseudogene.

In order to determine if such an endogenous antisense mRNA indeed existsfor the RbAp48 gene, a Northern blot analysis using sense and antisensesingle-stranded probes was performed. RNA from human A172 glioma cellsgrown under normal conditions or under hypoxia for 4 or 16 hours wereseparated by eleotrophosis on formaldehyde-agarose gels and blotted ontonylon membranes. One blot was hybridized with an antisense singlestranded probe which should reveal the existence of any antisense RNAand a second blot was hybridized with the antisense probe to reveal thenormal sense mRNA. As shown in FIG. 4, specific bands of different sizehybridized with the different probes. These results demonstrate theexistence of a mRNA species that hybridizes specifically with the senseprobe and which is of a different size from the normal sense mRNA andfurther indicate that an endogenous antisense mRNA indeed exists for theRbAp48 gene. Interestingly, an inverted correlation is observed in theexpression of the sense and antisense RbAp48 mRNAs under hypoxia, wherethe sense mRNA is reduced while the antisense mRNA is induced to higherlevels, suggesting a regulatory role for the antisense RNA.

As can be seen from Table 1, 14 of the 18 tested clones were found to bepositive under the experimental conditions or by bioinformatic analysis(clone 25-3-49). The bioinformatic analysis utilized the BLAST program(NCBI) to find matches between the clone and a certain region of anothergene that is complementary to it. The analysis thus showed that the“EASI” antisense enrichment methodology is efficient.

Table 2 identifies the inserts in the clones listed in Table 1 by SEQ IDNO.

TABLE 2 Insert from clone SEQ ID NO: N1_15 11 N1_27 12 N1_33 13 N2_20 14N2_66 15 N3_13 16 D1-1 17 D1-19 18 D1-48 19 D2-31 20 D2-3 21 D2-7 22D2-20 23 N26-3-50 24 N2-3-40 25 N3-45 26 25-3-49 27 20-2-33 28

EXAMPLE 2

To produce probes for microarray analysis from the EASI enriched cDNA,the following procedure may be used (graphically represented in FIG. 5)RNA is derived from cell population A (A-RNA) and from cell population B(B-RNA). A reverse transcription reaction with regular oligo-dT is usedto make single stranded cDNA from A-RNA (A-sscDNA). For B-RNA anoligo-dT that has an additional sequence of the T7 RNA polymerasepromoter region is used (e.g., SEQ ID NO:29). This produces a singlestranded cDNA population (B-sscDNA) that has the T7 RNA polymerasepromoter (T7RPP) region at its 5′ end. Using the regular EASI methoddescribed in Example 1, the A-sscDNA and B-sscDNA populations are mixed,and allowed to anneal to each other. A DNA polymerase that also has a 3′to 5′ exonuclease activity is then used (e.g., T4 DNA polymerase) toenrich for the population of cDNA that annealed to each other. Theresult of this reaction is a chimeric double stranded cDNA that has theT7 RPP on one of its ends. This now becomes the template for a T7 RNApolymerase reaction. The RNA produced from this reaction can now belabeled with a fluorescent dye for DNA microarray analysis usingstandard methods.

It should be noted that only in the cases where an antisense RNA isover-expressed in one population (e.g., A-RNA), and its complementarysense mRNA is over-expressed in the second population (e.g., B-RNA),will the T7RPP be on only one end of the double stranded cDNA template.If sense and antisense RNAs are found in the same population (B-RNA),they will anneal to each other and the resulting double stranded cDNAwill have the T7 RNA polymerase promoter region on both ends. Such atemplate will cause production of both sense and antisense RNA that willcancel each other during hybridization. Thus, significant labeling isproduced only in cases where there is differential expression of theantisense RNAs.

For hybridization to DNA microarrays, the EASI T7RP derived RNA can belabeled with one fluorescent die (e.g., Cy3). This probe can be comparedto a probe derived from A-RNA or from B-RNA. Such a probe will belabeled with another fluorescent dye (e.g., Cy5). After hybridization tothe DNA microarray, differentially expressed antisense RNA can bedetected by simple analysis of the hybridization results. Since the EASIT7RP derived probe enriches for cases in which there is differentialexpression of antisense RNA, the enrichment will be observed by a highersignal from the Cy3 label when compared to the Cy5 label.

One limitation of this approach is that the analysis cannot disclose thepattern of differential expression, namely, in which population is senseover-expressed and in which is antisense over-expressed. The followingprovides one way to circumvent this limitation. After the production ofsscDNA from B-RNA, the 3′ end is blocked by the addition of a nucleotideanalog such as deoxynucleoside [1-thio] triphosphate. Such an analog canbe added by using the enzyme terminal deoxy transferase (TdT) or byligating a primer that includes deoxynucleoside [1-thio] triphosphateanalogs, to the 3′ end with an appropriate ligase (e.g., T4 RNA ligase)that also ligates oligonucleotides to single stranded DNA. Anotheroption is to incorporate such analogs into the sscDNA during the reversetranscription reaction. Once such modified sscDNA, carrying the T7RPP onits 5′ end, is produced, it will be resistant to the exonucleaseactivity of the T4 DNA polymerase during the subsequent EASI reaction.Using this modification approach only sscDNA templates which areunmodified, coming from the A-RNA population, will be templates for theEASI procedure. Hybrids of sense and antisense cDNA derived from B-RNAwill be completely resistant to the EASI procedure and will not berepresented in the enriched population of EASI cDNA (FIG. 7). The onlypopulation of cDNA that will be meaningful is the one that has the T7RPPon one side (FIG. 6).

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by references.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. In is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

References

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1. A method for detection of differential expression of naturalantisense messenger RNA (mRNA), comprising: (a) separately obtainingpolyA-mRNA-A molecules from cell population A and polyA-mRNA-B moleculesfrom cell population B; (b) separately generating by a reversetranscription enzyme a population of single-stranded cDNA-A moleculesfrom polyA-mRNA-A and a population of single-stranded cDNA-B moleculesfrom polyA-mRNA-B, wherein the polydeoxythymidine containingoligonucleotide primer used to produce the cDNA-B molecules comprises aspecific bacteriophage RNA polymerase promoter region close to its 5′terminus; (c) incubating the combined populations of single-strandedcDNA-A molecules and single-stranded cDNA-B molecules, under conditionsallowing hybridization of sense cDNA molecules with antisense cDNAmolecules, wherein each single-stranded antisense cDNA molecule thathybridizes has a segment complementary to the sense cDNA molecule andhybridizes thereto to form a hybrid molecule with a double-strandedsegment; (d) treating the hybrid molecules with a DNA polymerase havinga 5′ to 3′ polymerase activity and a 3′ to 5′ exonuclease activity toremove single-stranded non-hybridized segments of the hybrid moleculefrom 3′ to 5′ and to extend the double-stranded segment of the hybridmolecule 5′ to 3′ over an adjacent single-stranded segment as template,thereby forming a double-stranded molecule having the RNA polymerasepromoter region close to one terminus; (e) using the double-strandedmolecule as a template for the specific RNA polymerase to a populationof RNA molecules; (f) labeling with a first label the RNA moleculesproduced in step (e); (g) labeling with a second label as control thepolyA-mRNA-A molecules and/or the polyA-mRNA-B molecules of step (a);(h) mixing labeled RNA molecules from steps (f) and (g) and hybridizingthem to a DNA microarray, and (i) identifying the genes on themicroarray which are preferentially labeled with the labeled RNAmolecules of step (f), wherein the genes so identified on the microarrayare detected as differentially expressed natural antisense mRNA.
 2. Themethod according to claim 1, wherein the specific bacteriophagepolymerase is selected from the group consisting of T7 RNA polymerase,T3 RNA polymerase, and SP6 RNA polymerase.
 3. The method according toclaim 1, wherein following step (b) the cDNA-B is modified in order toresist the 3′ to 5′ exonuclease activity of step (d).
 4. The methodaccording to claim 3, wherein in step (d) the 3′ terminus of the cDNA-Bis modified.
 5. The method according to claim 3, wherein in step (d) theentire cDNA-B is modified.
 6. The method according to claim 4, wherein3′ terminus of the cDNA-B is modified by addition of a nucleotideanalog.
 7. The method according to claim 5, wherein the entire cDNA-B ismodified by incorporation of nucleotide analogs.
 8. The method accordingto claim 1, wherein the first label of step (f) is Cy3, and the secondlabel of step (g) is Cy5.
 9. The method according to claim 1, whereinthe first label of step (f) is Cy5, and the second label of step (g) isCy3.
 10. A method for detection of differential expression of naturalantisense messenger RNA (mRNA), comprising: (a) separately obtainingpolyA-mRNA-A molecules from cell population A and polyA-mRNA-B moleculesfrom cell population B; (b) separately generating by a reversetranscription enzyme a population of single-stranded cDNA-A moleculesfrom polyA-mRNA-A and a population of single-stranded cDNA-B moleculesfrom polyA-mRNA-B, wherein the polydeoxythymidine containingoligonucleotide primer used to produce the cDNA-A molecules comprisesclose to its 5′ terminus a sequence identical to an amplification primerused in step (e) and wherein the polydeoxythymidine containingoligonucleotide primer used to produce the cDNA-B molecules comprises aspecific bacteriophage RNA polymerase promoter region close to its 5′terminus; (c) incubating the combined populations of single-strandedcDNA-A molecules and single-stranded cDNA-B molecules, under conditionsallowing hybridization of sense cDNA molecules with antisense cDNAmolecules, wherein each single-stranded antisense cDNA molecule thathybridizes has a segment complementary to the sense DNA molecule andhybridizes thereto to form a hybrid molecule with a double-strandedsegment; (d) treating the hybrid molecules with a DNA polymerase havinga 5′ to 3′ polymerase activity and a 3′ to 5′ exonuclease activity toremove single-stranded non-hybridized segments of the hybrid moleculefrom 3′ to 5′ and to extend the double-stranded segment of the hybridmolecule 5′ to 3′ over an adjacent single-stranded segment as template,thereby forming a double-stranded molecule having the RNA polymerasepromoter region close to one terminus; (e) amplifying thedouble-stranded molecule of step (d) using a thermostable polymerase anda first amplification primer identical to the sequence used in step (b)and a second amplification primer identical to the specificbacteriophage RNA polymerase promoter region of step (b); (f) using thedouble-stranded molecules so produced as a template for the specific RNApolymerase to produce a population of RNA molecules; (g) labeling with afirst label the RNA molecules produced in step (f); (h) labeling with asecond label as control the polyA-mRNA-A molecules and/or thepolyA-mRNA-B molecules of step (a); (i) mixing labeled RNA moleculesfrom steps (g) and (h) and hybridizing them to a DNA microarray; and (j)identifying the genes on the microarray which are preferentially labeledwith the labeled RNA molecules of step (g).
 11. The method according toclaim 10, wherein the specific bacteriophage polymerase is selected fromthe group consisting of T7 RNA polymerase, T3 RNA polymerase, and SP6RNA polymerase.
 12. The method according to claim 10, wherein followingstep (b) the cDNA-B is modified in order to resist the 3′ to 5′exonuclease activity of step (d).
 13. The method according to claim 12,wherein in step (d) the 3′ terminus of the cDNA-B is modified.
 14. Themethod according to claim 12, wherein in step (d) the entire cDNA-B ismodified.
 15. The method according to claim 13, wherein 3′ terminus ofthe cDNA-B is modified by addition of a nucleotide analog.
 16. Themethod according to claim 14, wherein the entire cDNA-R is modified byincorporation of nucleotide analogs.
 17. The method according to claim10, wherein the first label of step (g) is Cy3, and the second label ofstep (h) is Cy5.
 18. The method according to claim 1, wherein the firstlabel of step (g) is Cy5, and the second label of step (h) is Cy3.